Cancers or oncoses are, after cardiovascular diseases, the second most common cause of death in Germany. If therapy is started in good time or the oncosis occurs late in life and then only progresses slowly, not every cancer proves fatal. The current rate of cure for all cancers averages 30 to 40%, but there are marked variations depending on the actual cancer. For example, cancers of the respiratory tract, especially lung cancer, are among the poorly treatable cancers.
In cancers or in tumor cells, the coordination of growth, division and destruction or apoptosis in the cell cluster is disturbed or disabled. Often endogenous regulatory signals are not recognized or are not executed or are executed incorrectly, which is often linked causally to genetic defects or gene modifications, such as mutations. Genetic changes, such as mutations, can thus lead to changes in the structure and in the physiology of proteins encoded by the affected genes, which may induce or promote tumor growth. The development of cancer, or carcinogenesis, in particular the primary disease event, may thus be due to a change of the genetic material, which cannot be compensated by endogenous monitoring and correcting systems and consequently, for example, in the context of cell division processes, can be transmitted to succeeding cells, which sometimes leads to the development of a primary tumor.
Lung carcinomas, which are also designated with synonyms such as bronchial carcinomas, bronchogenic carcinoma or lung cancer, constitute a malignant oncosis based on degenerated cells in particular of the bronchi or bronchioles. Bronchial or lung cancer is one of the commonest malignant cancers in humans and represents one of the commonest causes of death due to cancer in the Western Hemisphere. The number of new cases of lung cancer in Germany is about 50,000 persons per year. The main cause of lung cancers is inhalation of cigarette smoke. In addition, there are some toxic substances, such as asbestos or chromium, that can also induce lung carcinomas. Owing to the sometimes completely absent or only nonspecific symptoms in the early stages of the disease, most first diagnoses of lung cancer are not made until the later stages of the disease, so that one of the most promising treatment options—complete surgical removal of the tumor—is often no longer possible, in particular also because metastasis has already begun. The rate of cure of bronchial carcinoma is generally very poor and a five-year survival rate is below 10%; the probability of survival after two years is less than 20%.
About a quarter of all malignant tumors or malignant neoplasms are bronchial carcinomas. In men, bronchial carcinoma is globally the commonest oncosis, in Germany it is the third-commonest after prostate cancer and colorectal carcinoma, but bronchial carcinoma is in first place as the cause of cancer deaths.
Based on their histology and the disease course, lung cancers are generally divided into two groups, namely small-cell lung cancer (SCLC) on the one hand and non-small-cell lung cancer (NSCLC) on the other hand. Non-small-cell lung cancer or NSCLC represents the largest group of bronchial carcinomas, with an incidence of 85% of lung cancers. Depending on the histological findings, non-small-cell lung cancer or NSCLC may be differentiated into a sometimes fusiform squamous cell carcinoma, an adenocarcinoma and a large-cell carcinoma or giant-cell carcinoma.
Regarding the therapeutic approaches known in the prior art for the treatment of lung cancer, in particular small-cell lung cancer, these focus primarily on a therapeutic approach based on chemotherapy or radiotherapy. However, therapies of this kind are associated with severe side-effects and often do not lead to the desired therapeutic success. In studies, even platinum-based combination therapies only achieve a median survival increase of just 10 to 12 months. Recently, patients with a diagnosis of small-cell lung cancer or NSCLC have been offered alternative therapies to the usual treatment with chemotherapeutics. Drugs are thus used which, in contrast to cytostatics, act specifically on tumor cells, and accordingly also have far fewer side effects. These include in particular the substances available under the international nonproprietary names gefitinib, erlotinib and cetuximab, which specifically bind to or inactivate the receptor of the epidermal growth factor (EGF) often involved in lung cancers, namely the so-called EGF receptor or EGFR.
The EGF receptor (epidermal growth factor receptor) is a member of the so-called ErbB family with a subfamily of four closely-related receptor tyrosine kinases. The EGF receptor is often also designated synonymously as HER1, EGFR1 or ErbB-1.
The EGF receptor is a transmembrane receptor with intrinsic tyrosine kinase activity, which occurs in all cell types. The receptor has a membrane channel and in the cytoplasmic portion it has a kinase domain with ATP binding site. The EGF receptor is classified among the growth factor receptors.
In non-malignant cells, after binding of its ligand (EGF), the receptor is activated by dimerization and phosphorylation and consequently conveys growth and survival signals into the interior of the cell. Activation of the receptor finally leads to stimulation of cell growth and prevention of apoptosis or programmed cell death. The EGF receptor supports proliferation and cell survival.
However, overexpression and/or certain mutations in the EGF receptor, such as are sometimes observed in tumor cells, bring about permanent or excessive activation of the receptor, which is accompanied by an undesirable level of cell growth, excessive cell division and therefore tumor formation or tumor growth. For malignant cells, constant imparting of growth signals is of advantage as they bring about or support the accelerated proliferation and survival of the malignant cells. Tumor cells that possess overexpression or activating mutations with respect to the EGF receptor are even dependent on the permanent or excessive activation of the EGF receptor for their proliferation and their survival. In various types of tumors, the EGF receptor is therefore up-regulated or is in a mutated form, which has the result that the tumor cells in question grow uncontrollably and multiply. The aforementioned active substances aim to block the oncogenic signal of the EGF receptor and thus stop or slow down tumor growth.
The EGF receptor may thus be directly linked to an oncosis or cancer, in particular a lung or bronchial carcinoma, such as small-cell lung cancer, especially as, in its mutated form, the EGF receptor leads to uncontrolled growth and multiplication of tumor cells. Specific blocking or inactivation of the, in particular mutated, EGF receptor can, therefore, lead to restriction or stopping of growth of tumor cells.
In the context of the present invention, it is important that through targeted inhibition of the EGF receptor, the activation of the receptor can be reduced or inhibited. Over 80% of the mutations of the EGF receptor in patients with small-cell lung cancer or NSCLC are based on various deletions in exon 19 of the EGF receptor and on a point mutation in exon 21, namely the so-called L858R mutation (i.e., exchange of the amino acid leucine L at position 858 in the amino acid sequence of the EGF receptor for the amino acid arginine R). Patients with a lung tumor who have one of these changes are especially suitable for therapy with EGF receptor inhibitors. In particular, the drugs or substances gefitinib and erlotinib have high specificity of action with respect to EGF receptors that have said mutations. Therapy with specific inhibitors of the EGF receptor, especially with respect to its mutated form, is generally well-tolerated and also displays a certain efficacy. Owing to the high specificity, the mutation-bearing receptors are inhibited selectively, which reduces side-effects and increases the therapeutic effect.
After a certain time, most patients develop a so-called secondary mutation, which arises in addition to the mutation already present and leads to resistance to erlotinib and gefitinib. In roughly 65% of these cases, a mutation is found in exon 20 of the EGF receptor, which is a T790M mutation (i.e., exchange of the amino acid threonine T for methionine M at position 790 of the EGF receptor). For these patients, drugs are available whose mechanism of action and specificity differ from the drugs of the so-called first generation, such as erlotinib and gefitinib. The inhibitors of the so-called second generation bind in particular irreversibly to the receptor, and not reversibly, as is the case with the aforementioned first-generation drugs. Patients with small-cell lung cancer, who, owing to the secondary mutation, in particular the T790M mutation, no longer respond to first-generation drugs, can therefore continue treatment with a second-generation EGF receptor inhibitor. These inhibitors are also highly specific and effective so that the growth and survival of the tumor cells can be slowed or prevented.
Against this technical and medical background, a rapid, easily managed mutation analysis that leads to exact results with respect to the EGF receptor in patients with lung cancer is therefore extremely important, in particular also against the background of tuning or optimizing the therapy with respect to the specific mutation finding.
In particular, to provide an optimum therapeutic approach by means of highly effective, individualized medicine, it is necessary to investigate the tumor tissue for the status of the EGF receptor, especially with respect to mutations that may be present, in particular as described above. On this basis, these patients can be treated with the corresponding EGF receptor inhibitors according to their mutation status.
Based on a highly informative mutation analysis with respect to the EGF receptor, it is then possible to carry out appropriate targeted therapy with the respective drugs.
Various methods or approaches based on molecular biology are available in the prior art for detecting mutations in genomic DNA from tumor tissue. For example, sequencing according to Sanger is used routinely. However, this method has the disadvantage that mutations can only be detected when the DNA bearing them is present at a level of at least 20% to 25% in the sample to be analyzed relative to the total DNA content of the sample. The expenditure of time for execution and evaluation is moreover relatively high, as the test can take several hours.
Another method of the prior art for the analysis of mutations is the so-called polymerase chain reaction (PCR), for example, so-called real-time PCR or RT-PCR. The analysis time can be reduced using this method. Moreover, execution is relatively economical and the sensitivity with respect to the mutation to be detected or analyzed is already higher. The results obtained with conventional PCR are nevertheless not always satisfactory, especially if the sample only has extremely small amounts of mutation material. As a result, conventional PCR only has a low level of sensitivity.